JP4430668B2 - Method for producing pectin - Google Patents

Description

  The present invention relates to a pectin obtained from a cell culture and a cosmetic containing the same. More specifically, the present invention relates to a pectin obtained from a cell culture of a Nigella plant and a high molecular weight pectin used as a moisturizing agent.

  Many organs of plants contain polysaccharides called pectin and are present in meristems and parenchyma. Pectin is a colloidal carbohydrate obtained from a plant body or from a plant body, and is a group of substances that contain a large number of galacturonic acid groups and are linked in a chain. The existence form is a form called a protopectin which binds to cellulose, hemicellulose, lignin, protein, mineral, etc. as a cell wall component of a plant and is insoluble in water. A substance that is water-soluble in pectin and gels with sugar and acid under appropriate conditions is called pectin.

The general production of pectin consists of the steps of (1) extraction under high temperature acidity from plant starting materials, (2) purification of the liquid extract, and (3) isolation of the extracted pectin from the liquid. Among them, in the acid extraction stage, the plant material is treated with a dilute acid such as nitric acid, sulfuric acid, hydrochloric acid or other inorganic or organic acids, usually under conditions of pH 1-2 and temperature 80 ° C-100 ° C. To do. Commonly used plant starting materials are citrus peel obtained from juice production and apple pomace obtained from apple juice and cider production, but the molecular weight of pectin obtained from these plant starting materials is 6 Only × 10 ^{4 to} 2 × 10 ⁵ .

It is known that pectin can be obtained by culturing plant cells in a liquid medium. Proc. Natl. Acad. Sci. USA, vol. 85, p. 2618-2622 (1988), it is reported that pectin having a molecular weight of 1.8 × 10 ⁴ is released from a cultured soybean cell into a liquid. 1 and a little. Moreover, woody plants such as the above-mentioned citrus fruits have a high content of phenolic substances in the cell walls and are hard tissues, so that tissue culture is difficult and is not suitable for producing pectin. Therefore, a plant that can produce a large amount of pectin by culturing is not known.

  WO 9636693 discloses that pectin fragments can be obtained by stimulating many plant cells with a DNA methylation inhibitor. However, this publication does not disclose any molecular weight or production amount of the obtained pectin.

  Pectin has various properties depending on the degree of esterification and molecular weight, and most of them are used in the food field as gelling agents, thickeners, stabilizers and the like. The degree of esterification (DE) indicating the degree of esterification is a numerical value (%) indicating the proportion of esterified galacturonic acid (carboxyl group) in the total galacturonic acid (total carboxyl group), and the degree of methyl esterification It is classified into high methoxyl pectin (HM pectin) and low methoxyl pectin (LM pectin). Usually, those having a DE higher than 50% are called HM pectin and those having a DE of 50% or less are called LM pectin. In general, most of the pectin extracted from plant starting materials under high-temperature acidic conditions is HM pectin. In order to obtain LM pectin, it is necessary to further demethylate using acid, alkali, enzyme or ammonia. There is. When extracting pectin from citrus peel, sugar chains were cleaved along with hydrolysis of esters due to high temperature acidic conditions and demethylation treatment during extraction, leading to a decrease in molecular weight. As a result, high molecular weight pectin is expected to have various properties, but a method for stably producing such pectin has not been known.

The use of pectin as a component of cosmetics is already known. Pectins used in cosmetics are exclusively pectin having a molecular weight of 6 × 10 ⁴ to 2 × 10 ⁵ , and they are added as gelling agents, thickeners, stabilizers, and as supplements to active cosmetic ingredients. However, it is not known to use pectin as an active ingredient of a humectant.
WO9636693 Y. Hayashi, K .; Yoshida, Proc. Natl. Acad. Sci. USA, vol. 85, p. 2618-2622 (1988)

As described above, the molecular weight of commercially available pectin obtained by extraction from conventional plant tissue is 6 × 10 ⁴ to 2 × 10 ⁵ . The problem to be solved by the invention is to find a characteristic of pectin having a higher molecular weight and to provide a method capable of supplying a large amount of pectin having a higher molecular weight.

In order to solve the above-mentioned problems, the present inventors have studied pectin produced by various plants in order to obtain a pectin having a higher molecular weight than conventional pectin, and intensively studied a method for producing the pectin in large quantities. As a result, it has been found that by inducing callus using a Nigella genus plant as a starting plant and further culturing cells, a higher molecular weight pectin can be obtained and a large amount of pectin can be produced compared to conventional commercially available pectin. The obtained higher molecular weight pectin was found to have excellent water retention ability and can be used as a humectant. The present invention has been completed based on these findings. That is, the present invention is as follows.
[1] Containing 70 to 90 mol% of galacturonic acid as a constituent sugar, the molecular weight range measured by gel filtration chromatography is 1 × 10 ^{4 to} 5 × 10 ⁷ , and the molecular weight showing a peak is 2 × A method for producing pectin, comprising obtaining and recovering pectin in the range of 10 ⁵ to 1 × 10 ⁷ from a culture or culture solution obtained by culturing cells of the genus Nigella.
[2] The molar ratio of the constituent sugars of the pectin is galacturonic acid: arabinose: galactose: glucuronic acid: glucose: xylose: rhamnose: mannose: fructose = 7-9: 0.4-0.7: 0.4-0 .6: 0.1 to 0.6: 0.05 to 0.5: 0.04 to 0.4: 0 to 0.1: 0 to 0.08: 0 to 0.05, The method for producing pectin according to [1].
[3] The method for producing pectin according to [1] or [2], wherein the plant belonging to the genus Nigella is Nigella damascena or Nigella sativa.
[4] Containing 70 to 90 mol% of galacturonic acid as a constituent sugar, the molecular weight range measured by gel filtration chromatography is 1 × 10 ^{4 to} 5 × 10 ⁷ , and the molecular weight showing a peak is 2 × A step of separating and collecting a pectin in the range of 10 ⁵ to 1 × 10 ⁷ from a culture or a culture solution obtained by culturing cells of the genus Nigella,
A method for producing a humectant comprising blending the pectin separated and recovered from the culture or the culture as an active ingredient .
[5] Containing 70 to 90 mol% of galacturonic acid as a constituent sugar, the molecular weight range measured by gel filtration chromatography is 1 × 10 ^{4 to} 5 × 10 ⁷ , and the molecular weight exhibiting a peak is 2 × A step of separating and collecting a pectin in the range of 10 ⁵ to 1 × 10 ⁷ from a culture or a culture solution obtained by culturing cells of the genus Nigella,
A method for producing a cosmetic comprising blending the pectin separated and recovered from the culture or the culture solution as an active ingredient of a moisturizing agent .

  The plant cell-derived pectin of the present invention has a higher molecular weight than the commercially available pectin obtained by the conventional method. Therefore, the pectin of the present invention is excellent in water retention ability. In addition, the gel strength is excellent and the viscosity is low. When the pectin of the present invention is used as a cosmetic material, it exhibits a good feeling of use and is optimal as a moisturizing agent for cosmetics.

It is a figure (graph) which shows the result of Example 12 (2). In the figure, A represents a molecular weight range and B represents a molecular weight peak. 10 is a diagram (graph) showing the results of Test Example 2. FIG. In the figure, triangles indicate pectin of the present invention, squares indicate sodium hyaluronate, circles indicate commercially available pectin, and X indicates water.

  The pectin of the present invention is a pectin obtained by separating and recovering from a culture or culture solution obtained by culturing plant cells. Preferably, it is a pectin obtained by separating and recovering from a culture or culture solution obtained by culturing cells of the genus Nigella.

  Nigella genus plants belong to the family Ranunculaceae, and there are about 20 species originating from the Mediterranean, West Asia and North Africa. Representative species are Nigella damascena, Nigella sativa, Nigella hispanica and Nigella arvensis. In the present invention, any plant of the genus Nigella may be used, but it is preferable to use Nigella damascena or Nigella sativa.

The pectin of the present invention can be produced by a two-stage culture that induces callus (dedifferentiated cells) and further propagates the obtained callus.
First, in the culture | cultivation which induces callus, tissue, such as a seed of Nigella genus plant, a leaf, a stem, and a root, 30-95% ethanol, 0.01-0.1% benzalkonium chloride, 0.1-5% order The surface of the plant body is sterilized with sodium chlorite to induce callus and culture. The callus-inducing medium is made by adding a carbon source and a plant hormone to medium components commonly used for plant tissue culture, such as Murashige Skoog, Rinsmeier Skoog, White, Niche, Gamborg, WPM (Woody Plant Medium). And sterilized by steam heating at 121 ° C. for 15 minutes. The carbon source is monosaccharides such as glucose and fructose or disaccharides and oligosaccharides such as sucrose, maltose and the like, which are used as constituent sugars. It is preferably used in the range of 5 to 5%. As plant hormones, auxins, cytokinins, gibberellins, abscisic acid, brassinosteroids and the like are used alone or in combination in a concentration range of 0 to 10 ⁻⁴ M. Examples of auxins include indoleacetic acid, indolebutyric acid, α-naphthaleneacetic acid, 2,4-dichlorophenoxyacetic acid, 2,6-dichlorobenzoic acid, and examples of cytokinins include zeatin, furfurylaminopurine (kinetine), benzyladenine, iso Examples include pentenyl adenine and dimethylaminopurine. The gibberellins are preferably active forms GA ₁ , GA ₃ , GA ₄ and GA ₇ , and the brassinosteroid is preferably brassinolide. Among the components added to the medium used for callus induction, substances that decompose by heating are added after sterilization by filtration using a 0.2 μm filter after the other components are sterilized by steam heat. The pH of the medium is adjusted to pH 5 to 7, preferably 5.5 to 6, with a dilute alkaline solution such as sodium hydroxide or potassium hydroxide at the time of preparation.

  Callus induction can be either a solid medium or a liquid medium, but it is usually preferable to culture the medium on a solid medium solidified with 0.4-2% agar or 0.1-0.5% gellite. . Culturing is performed at a temperature of 15 to 30 ° C., preferably 23 to 27 ° C., and at this time, the culture may be performed under either light or dark conditions. Usually, callus can be obtained as a cell mass having a diameter of 1 to 30 mm after 5 to 60 days of culture.

  The callus is then grown by transplanting the callus to a new medium and culturing. The main culture may be either solid culture or liquid culture, but is preferably performed in liquid culture in consideration of economy and mass productivity. Culturing is carried out in the same medium as that used for inducing callus, or in a medium with modifications such as reducing the salt concentration. A medium with a modification that reduces the salt concentration is one or more of the main inorganic salts such as potassium nitrate, sodium nitrate, ammonium nitrate, sodium dihydrogen phosphate, calcium chloride, magnesium sulfate in the medium components. It is a medium reduced to such an extent that it does not affect the production of. Further, organic acids such as casamino acid known as known additives, nitrogen sources such as amino acids, coconut milk, yeast extract, and polypeptone can be added to the medium at a concentration of 0.01 to 10 g / l.

  The culture temperature, light conditions, and culture period of the culture for growing the callus are the same as the culture conditions at the time of callus induction, and cultured as a cell mass formed by several to several hundred cells by repeating the culture 2 to 5 times. Cells are obtained. Furthermore, by continuously growing cultured cells, pectin can be produced in a colloidal state or solubilized state in a culture or a culture solution. In order to produce pectin in large quantities, liquid culture is preferable, and the culture method is batch culture in which the whole culture and culture solution are collected every 5 to 30 days, and some culture and culture solution are collected every 5 to 30 days. Any method of semi-batch culture in which a fresh medium is added and cultured, and continuous culture in which the collection of the culture and culture solution and the addition of the fresh medium are always performed at a constant rate may be used. Although the initial cell density can be cultured in the range of 1 to 300 g / l, high density pectin can be produced in a short period of time by increasing the density. Oxygen supply during liquid culture can be performed by either reciprocating or swirling shaking, direct ventilation to the culture medium, or indirect method using a follow fiber. In order to improve the production amount of pectin, it is effective to increase the oxygen supply amount. For example, in swirling shaking culture, the number of shakes is set to 100 revolutions / minute or more and 200 revolutions / minute or less. It is preferable to increase the gas-liquid interface by setting the amount of the medium to 5% or more and 30% or less of the volume. In the case of directly ventilating the culture solution, the aeration rate per culture solution is 5% to 20% by volume per minute. Is preferable. Increasing the amount of oxygen supply is also effective in increasing the cell growth rate.

  Pectin is present in a colloidal state or a solubilized state in the culture or culture medium thus obtained.

  In the cultivation of plant cells, the production method for culturing cells of the genus Nigella plant has particularly high pectin production.

  In order to recover pectin from the culture or culture solution, the colloidal or solubilized pectin is first solubilized. In solubilization, dilute alkaline solution such as water, chelating agent, acidic buffer, carbonate is added to the culture and culture solution so that the pectin concentration is 3 g / l or less, and 0 to 40 ° C., preferably Pectin is solubilized under mild temperature conditions of 15-35 ° C. Examples of the chelating agent include oxalic acid, ammonium oxalate, polymerized phosphate (sodium hexametaphosphate or calgon of food additive), ethylenediaminetetraacetic acid or salts thereof. Examples of the acidic buffer include phosphoric acid and citric acid, and examples of the carbonate include sodium carbonate and potassium carbonate.

  After solubilizing pectin, solids such as cells are removed from the solution containing pectin by filtration or centrifugation. Thereafter, 0.5 to 5 times volume, preferably 1 to 3 times volume, of an organic solvent such as ethanol or acetone is added to precipitate and collect pectin. The pectin can be concentrated to an arbitrary concentration by dissolving the collected precipitate in water again. If necessary, a pectin having an arbitrary molecular weight can be obtained by removing low molecular weight components using a dialysis membrane and an ultrafiltration membrane capable of fractionation of an appropriate molecular weight. The concentrated pectin solution can be lyophilized to obtain pectin as a white or slightly yellow powder.

The pectin thus obtained is a pectin having a molecular weight range of 1 × 10 ^{4 to} 5 × 10 ⁷ and a peak molecular weight of 2 × 10 ⁵ to 1 × 10 ⁷ . More preferably, the pectin has a molecular weight range of 1 × 10 ⁵ to 1 × 10 ⁷ and a peak molecular weight of 1 × 10 ^{6 to} 5 × 10 ⁶ . The molecular weight of pectin as used in the present invention means the molecular weight measured by gel filtration chromatography. The range of molecular weight means the lower limit and the upper limit of the molecular weight distribution of pectin measured by gel filtration chromatography. The molecular weight showing a peak means the molecular weight at the highest frequency in the molecular weight distribution of pectin measured by gel filtration chromatography.

  The molecular weight of pectin is determined by comparing the distribution curve obtained as a result of the measurement of gel filtration chromatography and the distribution curve of the pectin obtained as a result of the measurement of gel filtration chromatography of a standard substance having a known molecular weight. In the present invention, gel filtration chromatography for measuring the molecular weight of pectin is measured under the following conditions.

  The measurement conditions of gel filtration chromatography for determining the molecular weight of the pectin of the present invention are as follows. In the following conditions, a differential refractometer used as a detector is a detector that detects a substance by a refractive index difference from a mobile phase, and here, RID-10A manufactured by Shimadzu Corporation or an equivalent product is used. .

Column: Tosoh TSKgelPW series or equivalent
(Inner diameter: 7.8mm x Length: 300mm)
Filler: Hydrophilic vinyl polymer gel Mobile phase: 0.1 M sodium nitrate aqueous solution Flow rate: 0.5 ml / min Temperature: 40 ° C
Sample concentration: 1 g / l aqueous solution Sample injection volume: 10 μl
Detector: Differential refractometer

The gel filtration chromatography of the sample containing pectin and the standard substance is measured under the conditions described above. Create a calibration curve using the measurement results of the standard substance. The molecular weight of pectin in a sample is obtained as a relative value in terms of a standard substance by applying the measurement result of the sample to the calibration curve.
The method for obtaining the molecular weight from the measured value is specifically as follows.
The standard substances used are polyethylene glycol (molecular weight: 2 × 10 ⁶ , reagent manufactured by Wako Pure Chemical Industries), polyethylene oxide (molecular weight: 1 × 10 ⁶ , 5 × 10 ⁵ , 2 × 10 ⁵ , 5 × 10 ⁴ , GL Science Reagents), pullulan (molecular weight: 1.6 × 10 ⁶ , 8 × 10 ⁵ , 4 × 10 ⁵ , 2 × 10 ⁵ , reagent manufactured by Showa Denko KK). At least three kinds of standard substances having different molecular weights are appropriately selected from the above standard substances.
When pectin gel filtration chromatography is measured under the above conditions, a distribution curve peculiar to pectin with respect to the retention time axis is obtained. Further, when gel filtration chromatography of a standard substance having a known molecular weight is measured under the same conditions, a distribution curve peculiar to the standard substance with respect to the retention time axis can be obtained. In this case, the retention time is synonymous with the elution amount of the mobile phase (eluent). In the distribution curve of the sample or standard material, the retention time at the position showing the peak is specified as the retention time unique to each material.
A calibration curve is created from the relationship between the retention time specific to the standard substance and the molecular weight of the standard substance, and the molecular weight of the pectin is determined by applying the retention time specific to the pectin of the sample to the calibration curve. When measuring a pectin having a molecular weight of 2 × 10 ⁶ or more or 5 × 10 ⁴ or less, the molecular weight of the pectin is determined by extrapolating or interpolating the calibration curve.

  Among the constituent sugars in pectin, uronic acid in which the hydroxymethyl group at the end of the carbon chain is oxidized to a carboxyl group can be colorimetrically determined (absorbed at 530 nm) by a known carbazole sulfate method. The pectin of the present invention contains 70-96 mol% uronic acid. The composition of the constituent sugars of pectin obtained in the present invention is neutral sugar and uronic acid which are liberated after acid hydrolysis with hydrochloric acid, sulfuric acid, acetic acid or trifluoroacetic acid for 30 minutes to 6 hours under conditions of 50 to 100 ° C. Can be determined by high-performance liquid chromatography. The pectin of the present invention is composed of galacturonic acid, arabinose, galactose, glucuronic acid, glucose, xylose, rhamnose, mannose, fructose, etc., and the molar ratio of main components is galacturonic acid: arabinose: galactose: glucuronic acid: glucose: xylose: Rhamnose: Mannose: Fructose = 7-9: 0.4-0.7: 0.4-0.6: 0.1-0.6: 0.05-0.5: 0.04-0.4: It is 0-0.1: 0-0.08: 0-0.05. That is, the pectin of the present invention is a pectin containing galacturonic acid in an amount of 70 to 90 mol% as a main constituent sugar.

  The degree of esterification of pectin can be examined by a method for quantifying saponified carboxyl groups. The degree of esterification of the pectin of the present invention is 0 to 60%.

  The pectin of the present invention is a pectin having a higher molecular weight than the commercially available pectin obtained in the past. A further feature of the pectin of the present invention is a pectin having a high water retention capacity compared to conventional commercially available pectin. Moreover, it is a pectin which has the property which the conventional commercially available pectin has, ie, the property which is water-soluble and gelatinizes in contact with bivalent or more metal salts, such as a calcium ion, saccharide | sugar, and an acid.

  The pectin of the present invention is characterized by a high molecular weight and a high water retention capacity compared to conventional commercially available pectin. Therefore, it can be used in cosmetics as an active ingredient of a humectant. The moisturizing agent here is a substance mainly blended in cosmetics in order to prevent moisture from evaporating from the skin and to provide moisture regulation and moist feeling on the skin surface. The pectin of the present invention is a sodium hyaluronate that is generally used as a moisturizing agent in any of the tests in the water retention test by the known paper disk method and the biological stratum corneum water load test by the skin stratum corneum moisture measuring device. It shows higher water retention capacity. Since the pectin of the present invention is particularly excellent in the initial moisture retention ability, it is excellent in skin permeability and affinity for the skin. In addition, the pectin of the present invention has a lower viscosity than sodium hyaluronate, although it has the same high molecular weight as sodium hyaluronate. Therefore, a feeling of moistness and smoothness is given to the user, and a feeling of use with little stickiness is given. The pectin of the present invention is a cosmetic material with excellent moistness and smoothness, such as a solution such as a lotion, a gel such as a cleansing foam, an emulsion such as an emulsion, a solid such as a soap and a lipstick, Various cosmetics having a powder form such as a press powder, a paste form such as a foundation, a film form such as a pack, and an aerosol form such as a hair spray can be provided.

  In addition to the pectin of the present invention, the cosmetics of the present invention include components usually used in cosmetics and pharmaceuticals, for example, powder components, liquid fats, solid fats, waxes, hydrocarbon oils, higher fatty acids, ester oils, silicone oils. , Anionic surfactants, cationic surfactants, amphoteric surfactants, nonionic surfactants, water-soluble polymers, thickeners, UV absorbers, sequestering agents, alcohols, sugars, amino acids, organic amines, Polymer emulsion, pH adjuster, vitamins, antioxidants, antioxidant aids, water, etc. are blended as necessary, solution, gel, emulsion, solid, powder, paste, It can be produced by a generally known method according to the intended dosage form, such as a film-like or aerosol-like cosmetic. Specific ingredients that can be blended are listed below. The cosmetic of the present invention can be prepared by blending the pectin of the present invention and any one or more of the following components.

  Examples of the powder component include inorganic powders (for example, talc, kaolin, mica, sericite (sericite), muscovite, phlogopite, synthetic mica, saucite, biotite, permiculite, magnesium carbonate, calcium carbonate, silicic acid. Aluminum, barium silicate, calcium silicate, magnesium silicate, strontium silicate, metal tungstate, magnesium, silica, zeolite, barium sulfate, calcined calcium sulfate (baked gypsum), calcium phosphate, fluorine apatite, hydroxyapatite, ceramic powder Metal soap (eg, zinc myristate, calcium palmitate, aluminum stearate), boron nitride, etc .; organic powder (eg, polyamide resin powder (nylon powder), polyethylene powder, polymethyl methacrylate powder, Styrene powder, copolymer resin powder of styrene and acrylic acid, benzoguanamine resin powder, polytetrafluoroethylene powder, cellulose powder, etc.); inorganic white pigment (eg, titanium dioxide, zinc oxide, etc.); inorganic red pigment (eg, , Iron oxide (Bengara), iron titanate, etc.); inorganic brown pigment (for example, γ-iron oxide, etc.); inorganic yellow pigment (for example, yellow iron oxide, loess); inorganic black pigment (for example, black) Iron oxide, low-order titanium oxide, etc.); inorganic purple pigments (eg, manganese violet, cobalt violet, etc.); inorganic green pigments (eg, chromium oxide, chromium hydroxide, cobalt titanate, etc.); inorganic blue pigments (eg, For example, ultramarine blue, bitumen, etc.); pearl pigments (eg, titanium oxide coated mica, titanium oxide coated bismuth oxychloride, titanium oxide coated titanium) Lac, colored titanium oxide coated mica, bismuth oxychloride, fish scale foil, etc.); metal powder pigments (eg, aluminum powder, copper powder, etc.); organic pigments such as zirconium, barium, or aluminum lake (eg, red 201, red 202) No., Red 204, Red 205, Red 220, Red 226, Red 228, Red 405, Orange 203, Orange 204, Yellow 205, Yellow 401, and Blue 404 Red No. 3, Red No. 104, Red No. 106, Red No. 227, Red No. 230, Red No. 401, Red No. 505, Orange No. 205, Yellow No. 4, Yellow No. 5, Yellow No. 202, Yellow No. 203, Green No. 3 and Blue No. 1); natural pigments (for example, chlorophyll, β-carotene, etc.) and the like.

  Examples of liquid oils include avocado oil, camellia oil, turtle oil, macadamia nut oil, corn oil, mink oil, olive oil, rapeseed oil, egg yolk oil, sesame oil, persic oil, wheat germ oil, southern castor oil, castor oil, linseed oil , Safflower oil, cottonseed oil, eno oil, soybean oil, peanut oil, tea seed oil, kaya oil, rice bran oil, cinnagiri oil, Japanese kiri oil, jojoba oil, germ oil, triglycerin and the like.

  Examples of the solid fat include cacao butter, coconut oil, hardened coconut oil, palm oil, palm kernel oil, owl kernel oil, hardened oil, owl, and hardened castor oil.

  Examples of waxes include beeswax, candelilla wax, cotton wax, carnauba wax, bayberry wax, ibota wax, whale wax, montan wax, nuka wax, lanolin, kapok wax, lanolin acetate, liquid lanolin, sugar cane wax, lanolin fatty acid isopropyl, hexyl laurate, reduced lanolin, Examples include jojoba wax, hard lanolin, shellac wax, POE lanolin alcohol ether, POE lanolin alcohol acetate, POE cholesterol ether, lanolin fatty acid polyethylene glycol, and POE hydrogenated lanolin alcohol ether.

  Examples of the hydrocarbon oil include liquid paraffin, ozokerite, squalane, pristane, paraffin, ceresin, squalene, petrolatum, microcrystalline wax, and the like.

  Examples of higher fatty acids include lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, oleic acid, undecylenic acid, tall oil fatty acid, isostearic acid, linoleic acid, linolenic acid, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) etc. are mentioned.

  Examples of ester oils include isopropyl myristate, cetyl octanoate, octyldodecyl myristate, isopropyl palmitate, butyl stearate, hexyl laurate, myristyl myristate, decyl oleate, hexyl decyl dimethyloctanoate, cetyl lactate, lactic acid Myristyl, lanolin acetate, isocetyl stearate, isocetyl isostearate, cholesteryl 12-hydroxystearate, ethylene glycol di-2-ethylhexanoate, dipentaerythritol fatty acid ester, monoisostearate N-alkyl glycol, neopentyl glycol dicaprate, Diisostearyl malate, glycerin di-2-heptylundecanoate, trimethylolpropane tri-2-ethylhexanoate, triiso Trimethylolpropane teaate, pentaerythritol tetra-2-ethylhexanoate, glycerin tri-2-ethylhexanoate, glyceryl trioctanoate, glyceryl triisopalmitate, trimethylolpropane triisostearate, cetyl 2-ethylhexanoate, 2-ethylhexyl palmitate, glyceryl trimyristate, tri-2-heptylundecanoic acid glyceride, castor oil fatty acid methyl ester, oleyl oleate, acetoglyceride, 2-heptylundecyl palmitate, diisobutyl adipate, N-lauroyl-L -Glutamic acid-2-octyldodecyl ester, di-2-heptylundecyl adipate, ethyl laurate, di-2-ethylhexyl sebacate, 2-hexyl myristate Rudeshiru, palmitic acid 2-hexyl decyl, 2-hexyldecyl adipate, diisopropyl sebacate, 2-ethylhexyl succinate, and triethyl citrate.

  Examples of the silicone oil include linear polysiloxanes (for example, dimethylpolysiloxane, methylphenylpolysiloxane, diphenylpolysiloxane, etc.); cyclic polysiloxanes (for example, octamethylcyclotetrasiloxane, decamethylcyclopentasiloxane, dodecamethylcyclohexyl). And silicone resins that form a three-dimensional network structure, various modified polysiloxanes (amino-modified polysiloxane, polyether-modified polysiloxane, alkyl-modified polysiloxane, fluorine-modified polysiloxane, etc.) It is done.

  Anionic surfactants include, for example, fatty acid soaps (eg, sodium laurate, sodium palmitate, etc.); higher alkyl sulfates (eg, sodium lauryl sulfate, potassium lauryl sulfate, etc.); alkyl ether sulfates (eg, POE-lauryl sulfate triethanolamine, POE-sodium lauryl sulfate, etc.); N-acyl sarcosine acid (eg, sodium lauroyl sarcosine, etc.); higher fatty acid amide sulfonates (eg, sodium N-myristoyl-N-methyl taurate, palm Oil fatty acid methyl taurine sodium, lauryl methyl taurine sodium, etc .; Phosphate ester salt (POE-oleyl ether sodium phosphate, POE-stearyl ether phosphate, etc.); Sulfosuccinate (for example, Sodium di-2-ethylhexyl sulfosuccinate, sodium monolauroyl monoethanolamide polyoxyethylene sodium sulfosuccinate, sodium lauryl polypropylene glycol sulfosuccinate, etc .; alkyl benzene sulfonates (eg, sodium linear dodecyl benzene sulfonate, triso linear dodecyl benzene sulfonate) Ethanolamine, linear dodecyl benzene sulfonic acid, etc.); higher fatty acid ester sulfates (for example, hydrogenated coconut oil fatty acid sodium glycerin sulfate, etc.); N-acyl glutamate (for example, monosodium N-lauroyl glutamate, di-N-stearoyl glutamate) Sodium, N-myristoyl-L-monosodium glutamate, etc.); sulfated oil (eg funnel oil); POE-A Kill ether carboxylic acid; POE-alkyl allyl ether carboxylate; α-olefin sulfonate; higher fatty acid ester sulfonate; secondary alcohol sulfate ester; higher fatty acid alkylolamide sulfate ester; lauroyl monoethanolamide succinic acid Sodium; N-palmitoyl aspartate ditriethanolamine; sodium caseinate and the like.

  Examples of the cationic surfactant include alkyltrimethylammonium salts (eg, stearyltrimethylammonium chloride, lauryltrimethylammonium chloride, etc.); alkylpyridinium salts (eg, cetylpyridinium chloride, etc.); distearyldimethylammonium dialkyldimethylammonium chloride; Poly (N, N′-dimethyl-3,5-methylenepiperidinium chloride); alkyl quaternary ammonium salt; alkyldimethylbenzylammonium salt; alkylisoquinolinium salt; dialkyl morpholinium salt; POE-alkylamine; Examples include alkylamine salts; polyamine fatty acid derivatives; amyl alcohol fatty acid derivatives; benzalkonium chloride; benzethonium chloride and the like.

  Examples of amphoteric surfactants include imidazoline-based amphoteric surfactants (eg, 2-undecyl-N, N, N- (hydroxyethylcarboxymethyl) -2-imidazoline sodium, 2-cocoyl-2-imidazolinium hydroxide). Side-1-carboxyethyloxy disodium salt, etc.); betaine surfactants (for example, 2-heptadecyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine, lauryldimethylaminoacetic acid betaine, alkylbetaine, amide betaine) , Sulfobetaine, etc.).

  Among the nonionic surfactants, lipophilic nonionic surfactants include, for example, sorbitan fatty acid esters (for example, sorbitan monooleate, sorbitan monoisostearate, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate). Rate, sorbitan sesquioleate, sorbitan trioleate, penta-2-ethylhexyl diglycerol sorbitan, tetra-2-ethylhexyl diglycerol sorbitan, etc .; glycerin polyglycerin fatty acids (eg mono cottonseed oil fatty acid glycerin, glyceryl monoerucate, Glyceryl sesquioleate, glyceryl monostearate, α, α'-oleic acid pyroglutamic acid glycerin, monostearic acid glycerin malic acid, etc.); propylene glycol fat Fatty acid esters (for example, propylene glycol monostearate); hardened castor oil derivative; glycerin alkyl ether and the like.

  Examples of hydrophilic nonionic surfactants among nonionic surfactants include POE-sorbitan fatty acid esters (for example, POE-sorbitan monooleate, POE-sorbitan monostearate, POE-sorbitan monooleate, POE-). Sorbitan tetraoleate, etc.); POE sorbite fatty acid esters (for example, POE-sorbite monolaurate, POE-sorbite monooleate, POE-sorbite pentaoleate, POE-sorbite monostearate, etc.); POE-glycerin fatty acid ester (For example, POE-glycerol monostearate, POE-glycerol monoisostearate, POE-monooleate such as POE-glycerol triisostearate, etc.); POE-fatty acid esters (eg PO -Distearate, POE-monodiolate, ethylene glycol distearate, etc.); POE-alkyl ethers (eg, POE-lauryl ether, POE-oleyl ether, POE-stearyl ether, POE-behenyl ether, POE-2-octyldodecyl ether, POE-cholestanol ether, etc.); Pluronic type (for example, Pluronic, etc.); POE • POP-alkyl ethers (for example, POE • POP-cetyl ether, POE • POP-2-decyltetradecyl ether, POE • POP−) Monobutyl ether, POE / POP-hydrogenated lanolin, POE / POP-glycerin ether, etc.); tetra-POE / tetra-POP-ethylenediamine condensates (for example, Tetronic, etc.); POE-Hima Oil-hardened castor oil derivatives (for example, POE-castor oil, POE-hardened castor oil, POE-hardened castor oil monoisostearate, POE-hardened castor oil triisostearate, POE-hardened castor oil monopyroglutamic acid monoisostearic acid Diester, POE-hardened castor oil maleic acid, etc.); POE-beeswax lanolin derivative (eg, POE-sorbit beeswax etc.); alkanolamide (eg, coconut oil fatty acid diethanolamide, lauric acid monoethanolamide, fatty acid isopropanolamide, etc.) POE-propylene glycol fatty acid ester; POE-alkylamine; POE-fatty acid amide; sucrose fatty acid ester; alkyl ethoxydimethylamine oxide; trioleyl phosphoric acid and the like.

  Among the water-soluble polymers, natural water-soluble polymers include, for example, plant-based polymers (for example, gum arabic, gum tragacanth, galactan, guar gum, carob gum, caraya gum, carrageenan, kanten, quince seed (malmello), alge colloid ( Cassava extract), starch (rice, corn, potato, wheat), glycyrrhizic acid); microbial polymer (eg, xanthan gum, dextran, succinoglucan, bullulan, etc.); animal polymer (eg, collagen, casein, albumin, Gelatin) and the like.

  Among the water-soluble polymers, as the semi-synthetic water-soluble polymer, for example, starch polymers (for example, carboxymethyl starch, methylhydroxypropyl starch, etc.); cellulose polymers (methylcellulose, ethylcellulose, methylhydroxypropylcellulose, Hydroxyethyl cellulose, sodium cellulose sulfate, hydroxypropyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose sodium, crystalline cellulose, cellulose powder and the like); alginic acid polymers (for example, sodium alginate, propylene glycol alginate, etc.) and the like.

  Among the water-soluble polymers, synthetic water-soluble polymers include, for example, vinyl polymers (eg, polyvinyl alcohol, polyvinyl methyl ether, polyvinyl pyrrolidone, carboxyvinyl polymer, etc.); polyoxyethylene polymers (eg, polyethylene). Glycol 20,000, 40,000, 60,000 polyoxyethylene polyoxypropylene copolymer, etc.); acrylic polymer (eg, sodium polyacrylate, polyethyl acrylate, polyacrylamide, etc.); polyethyleneimine; cation Examples thereof include polymers.

  Examples of thickeners include gum arabic, carrageenan, caraya gum, gum tragacanth, carob gum, quince seed (malmello), casein, dextrin, gelatin, sodium pectate, sodium alginate, methylcellulose, ethylcellulose, CMC, hydroxyethylcellulose, hydroxypropyl Cellulose, PVA, PVM, PVP, sodium polyacrylate, carboxyvinyl polymer, locust bean gum, guar gum, tamarind gum, cellulose dialkyldimethylammonium sulfate, xanthan gum, magnesium aluminum silicate, bentonite, hectorite, AlMg silicate (Beegum), Examples thereof include laponite and silicic anhydride.

Examples of the ultraviolet absorber include the following compounds.
(1) Benzoic acid-based ultraviolet absorbers such as paraaminobenzoic acid (hereinafter abbreviated as PABA), PABA monoglycerin ester, N, N-dipropoxy PABA ethyl ester, N, N-diethoxy PABA ethyl ester, N, N-dimethyl PABA ethyl ester, N, N-dimethyl PABA butyl ester, N, N-dimethyl PABA ethyl ester and the like.
(2) Anthranilic acid-based UV absorbers such as homomenthyl-N-acetylanthranilate.
(3) Salicylic acid ultraviolet absorbers such as amyl salicylate, menthyl salicylate, homomenthyl salicylate, octyl salicylate, phenyl salicylate, benzyl salicylate, p-isopropanol phenyl salicylate and the like.
(4) Cinnamic acid ultraviolet absorbers such as octyl cinnamate, ethyl-4-isopropyl cinnamate, methyl-2,5-diisopropyl cinnamate, ethyl-2,4-diisopropyl cinnamate, methyl-2,4- Diisopropyl cinnamate, propyl-p-methoxycinnamate, isopropyl-p-methoxycinnamate, isoamyl-p-methoxycinnamate, octyl-p-methoxycinnamate (2-ethylhexyl-p-methoxycinnamate), 2-ethoxy Ethyl-p-methoxycinnamate, cyclohexyl-p-methoxycinnamate, ethyl-α-cyano-β-phenylcinnamate, 2-ethylhexyl-α-cyano-β-phenylcinnamate, glyceryl mono-2-ethylhexanoyl -Diparamet Such as Shishin'nameto.
(5) Triazine-based ultraviolet absorbers such as bisresorcinyl triazine.
More specifically, bis {[4- (2-ethylhexyloxy) -2-hydroxy] phenyl} -6- (4-methoxyphenyl) 1,3,5-triazine, 2,4,6-tris { 4- (2-ethylhexyloxycarbonyl) anilino} 1,3,5-triazine and the like.
(6) Other ultraviolet absorbers such as 3- (4′-methylbenzylidene) -d, l-camphor, 3-benzylidene-d, l-camphor, 2-phenyl-5-methylbenzoxazole, 2,2 '-Hydroxy-5-methylphenylbenzotriazole, 2- (2'-hydroxy-5'-t-octylphenyl) benzotriazole, 2- (2'-hydroxy-5'-methylphenylbenzotriazole), dibenzalazine, dianisoyl Methane, 4-methoxy-4′-t-butyldibenzoylmethane, 5- (3,3-dimethyl-2-norbornylidene) -3-pentan-2-one, etc. Pyridazine derivatives such as dimorpholinopyridazinone.

  Examples of the sequestering agent include 1-hydroxyethane-1,1-diphosphonic acid, 1-hydroxyethane-1,1-diphosphonic acid tetrasodium salt, disodium edetate, trisodium edetate, and tetrasodium edetate. Sodium citrate, sodium polyphosphate, sodium metaphosphate, gluconic acid, phosphoric acid, citric acid, ascorbic acid, succinic acid, edetic acid, trisodium ethylenediaminehydroxyethyl triacetate and the like.

  Among the alcohols, examples of the lower alcohol include ethanol, propanol, isopropanol, isobutyl alcohol, and t-butyl alcohol.

  Among the alcohols, examples of the polyhydric alcohol include divalent alcohols (for example, ethylene glycol, propylene glycol, trimethylene glycol, 1,2-butylene glycol, 1,3-butylene glycol, tetramethylene glycol, 2,3 -Butylene glycol, pentamethylene glycol, 2-butene-1,4-diol, hexylene glycol, octylene glycol, etc.); trivalent alcohols (eg, glycerin, trimethylolpropane, etc.); tetravalent alcohols (eg, 1 , 2,6-hexanetriol, etc.); pentavalent alcohol (eg, xylitol, etc.); hexavalent alcohol (eg, sorbitol, mannitol, etc.); polyhydric alcohol polymer (eg, diethylene glycol, dipropylene, etc.) Glycol, triethylene glycol, polypropylene glycol, tetraethylene glycol, diglycerin, polyethylene glycol, triglycerin, tetraglycerin, polyglycerin, etc.); divalent alcohol alkyl ethers (eg, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether) , Ethylene glycol monobutyl ether, ethylene glycol monophenyl ether, ethylene glycol monohexyl ether, ethylene glycol mono 2-methylhexyl ether, ethylene glycol isoamyl ether, ethylene glycol benzyl ether, ethylene glycol isopropyl ether, ethylene glycol dimethyl ether, ethylene glycol diethyl ether , Ethylene Dihydric alcohol alkyl ethers (eg, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, diethylene glycol monobutyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, diethylene glycol butyl ether, diethylene glycol methyl ethyl ether, triethylene glycol monomethyl ether, triethylene glycol monomethyl ether, Ethylene glycol monoethyl ether, propylene glycol monomethyl ether, propylene glycol monoethyl ether, propylene glycol monobutyl ether, propylene glycol isopropyl ether, dipropylene glycol methyl ether, dipropylene glycol ethyl ether, Dipropylene glycol butyl ether, etc.); dihydric alcohol ether esters (for example, ethylene glycol monomethyl ether acetate, ethylene glycol monoethyl ether acetate, ethylene glycol monobutyl ether acetate, ethylene glycol monophenyl ether acetate, ethylene glycol diazebate, ethylene glycol di) Succinate, diethylene glycol monoethyl ether acetate, diethylene glycol monobutyl ether acetate, propylene glycol monomethyl ether acetate, propylene glycol monoethyl ether acetate, propylene glycol monopropyl ether acetate, propylene glycol monophenyl ether acetate); glycerin monoa Kill ethers (for example, chimyl alcohol, ceralkyl alcohol, batyl alcohol, etc.); sugar alcohols (for example, sorbitol, maltitol, maltotriose, mannitol, sucrose, erythritol, glucose, fructose, amylolytic sugar, maltose, xylitol Glycolide; tetrahydrofurfuryl alcohol; POE-tetrahydrofurfuryl alcohol; POP-butyl ether; POP-POE-butyl ether; tripolyoxypropylene glycerin ether; POP-glycerin ether; POP-glycerin Ether phosphoric acid; POP / POE-pentane erythritol ether, polyglycerin and the like.

  Examples of higher alcohols include linear alcohols (eg, lauryl alcohol, cetyl alcohol, stearyl alcohol, behenyl alcohol, myristyl alcohol, oleyl alcohol, cetostearyl alcohol); branched chain alcohols (eg, monostearyl glycerin ether (batyl alcohol) ), 2-decyltetradecinol, lanolin alcohol, cholesterol, phytosterol, hexyldodecanol, isostearyl alcohol, octyldodecanol and the like.

  Monosaccharides among saccharides include, for example, tricarbon sugars (eg, D-glyceryl aldehyde, dihydroxyacetone, etc.); tetracarbon sugars (eg, D-erythrose, D-erythrulose, D-treose, erythritol, etc.); Sugars (eg, L-arabinose, D-xylose, L-lyxose, D-arabinose, D-ribose, D-ribulose, D-xylulose, L-xylulose, etc.); hexoses (eg, D-glucose, D- Talose, D-bucicose, D-galactose, D-fructose, L-galactose, L-mannose, D-tagatose, etc.); pentose sugar (eg, aldheptose, heproose, etc.); octose sugar (eg, octulose, etc.); Deoxy sugars (eg 2-deoxy-D-ribose, 6-deoxy-L-galactose, 6-deoxy Amino sugar (eg, D-glucosamine, D-galactosamine, sialic acid, aminouronic acid, muramic acid, etc.); uronic acid (eg, D-glucuronic acid, D-mannuronic acid, L-guluron) Acid, D-galacturonic acid, L-iduronic acid, and the like).

  Among the saccharides, examples of the oligosaccharide include sucrose, umbelliferose, lactose, planteose, isoliquenose, α, α-trehalose, raffinose, lycnose, umbilicin, stachyose verbus course and the like.

  Examples of polysaccharides include saccharides such as cellulose, quince seed, chondroitin sulfate, starch, galactan, dermatan sulfate, glycogen, gum arabic, heparan sulfate, hyaluronic acid, tragacanth gum, keratan sulfate, chondroitin, xanthan gum, mucoitin sulfate, guar gum, dextran. , Keratosulfuric acid, locust bingham, succinoglucan, caronic acid and the like.

  Examples of amino acids include neutral amino acids (eg, threonine, cysteine, etc.); basic amino acids (eg, hydroxylysine, etc.) and the like. Examples of the amino acid derivative include acyl sarcosine sodium (lauroyl sarcosine sodium), acyl glutamate, acyl β-alanine sodium, glutathione, and pyrrolidone carboxylic acid.

  Examples of the organic amine include monoethanolamine, diethanolamine, triethanolamine, morpholine, triisopropanolamine, 2-amino-2-methyl-1,3-propanediol, 2-amino-2-methyl-1-propanol, and the like. Is mentioned.

  Examples of the polymer emulsion include an acrylic resin emulsion, a polyethyl acrylate emulsion, an acrylic resin liquid, a polyacryl alkyl ester emulsion, a polyvinyl acetate resin emulsion, and a natural rubber latex.

  Examples of the pH adjusting agent include buffers such as lactic acid-sodium lactate, citric acid-sodium citrate, and succinic acid-sodium succinate.

  Examples of vitamins include vitamins A, B1, B2, B6, C, E and derivatives thereof, pantothenic acid and derivatives thereof, biotin and the like.

  Examples of the antioxidant include tocopherols, dibutylhydroxytoluene, butylhydroxyanisole, gallic acid esters and the like.

  Examples of the antioxidant assistant include phosphoric acid, citric acid, ascorbic acid, maleic acid, malonic acid, succinic acid, fumaric acid, kephalin, hexametaphosphate, phytic acid, and ethylenediaminetetraacetic acid.

  The cosmetics of the present invention may be used in combination with the pectin of the present invention and other moisturizing agents. Examples of moisturizing agents that can be used in combination include polyethylene glycol, propylene glycol, glycerin, 1,3-butylene glycol, xylitol, Sorbitol, maltitol, chondroitin sulfate, hyaluronic acid, mucoitin sulfate, caronic acid, atelocollagen, cholesteryl-12-hydroxystearate, sodium lactate, bile salt, dl-pyrrolidone carboxylate, short chain soluble collagen, diglycerin (EO ) PO adduct, Izayoi rose extract, Achillea millefolium extract, Merirot extract and the like.

  Examples of other components that can be blended include preservatives (methyl paraben, ethyl paraben, butyl paraben, phenoxyethanol, etc.); anti-inflammatory agents (eg, glycyrrhizic acid derivatives, glycyrrhetinic acid derivatives, salicylic acid derivatives, hinokitiol, zinc oxide, allantoin, etc.); Whitening agents (eg, Yukishinoshita extract, arbutin, etc.); various extracts (eg, buckwheat, auren, shikon, peonies, assembly, birch, sage, loquat, carrot, aloe, mallow), iris, grape, yokuinin, loofah, lily , Saffron, nematode, ginger, hypericum, onionis, garlic, capsicum, chimney, red snapper, seaweed, etc., activator (eg, royal jelly, photosensitizer, cholesterol derivative, etc.); blood circulation promoter (eg, nicotinic acid) Benzyl ester, nicotinic acid β-butoxyethyl ester, capsaicin, gingerone, cantalis tincture, ictamol, tannic acid, α-borneol, nicotinic acid tocopherol, inositol hexanicotinate, cyclandrate, cinnarizine, trazoline, acetylcholine, verapamil, cephalanthin, anti-seborrheic agents (eg, sulfur, thianthol, etc.); anti-inflammatory agents (eg, tranexamic acid, thiotaurine, hypotaurine, etc.); herbal medicines (eg, calqueja (South America)), Palo Azul [palo azul] (South America)], Maseramista (Macelamista (South America))) and the like. A combination of the pectin of the present invention and one or more of the above components can also be provided as a multifunctional cosmetic.

  The pectin of the present invention is 0.0001 to 50% by weight, preferably 0.001 to 20% by weight, more preferably 0.01 to 10% by weight, and particularly preferably 0.02 to 10% by weight based on the total amount of the cosmetic. Blends weight percent. When the blending amount is less than 0.0001% by weight with respect to the cosmetic, the moisturizing ability is poor. Even when the blending amount exceeds 50% by weight, an increase in the effect cannot be expected.

  The cosmetic thus obtained is excellent in moisturizing ability, gives the user a moist and smooth feeling, and gives a feeling of use with little stickiness.

  In addition to the use as a moisturizer, the pectin of the present invention is used in, for example, jams and marmalade mucous substances, beverage stabilizers, pudding and cream gelling agents in the food field, and the pharmaceutical field. However, it can be used in the same manner as conventional commercially available pectin.

EXAMPLES Hereinafter, although this invention is demonstrated in more detail using an Example etc., this invention is not restrict | limited by these.
[Example 1]
The surface of Nigella damascena seedlings was sterilized with 70% ethanol, then treated with sodium hypochlorite (effective chlorine concentration 1%) for 15 minutes, and then washed three times with sterilized water. Sterilized water was prepared by steam autoclaving at 121 ° C. for 15 minutes. On a solid medium solidified with 0.8% agar of WPM medium (adjusted to pH 5.7 with 0.1 M sodium hydroxide) shown in Table 1 containing 3% sucrose and 10 ⁻⁵ Mα-naphthylacetic acid as a plant hormone Surface-sterilized black seedling seedlings were placed aseptically. After culturing at 25 ° C. in the dark for 1 week, amorphous dedifferentiated cells (callus) were induced. A part of the obtained callus was separated and placed on a solid medium having the same composition as the medium used for callus induction to grow. By repeating this process three times, a cell line that stably grew was obtained.

[Example 2]
The cells induced according to Example 1 were suspended in a liquid medium having the composition shown in Table 1 and cultured. The culture conditions were as follows: 300 ml of medium was added to a 3 l flask, 25 ° C., dark place, cell density 9.6 g / l, rotary shaking culture at 100 rpm, and culture period 3 weeks. The chelating agent CyDTA (trans-1,2-diaminocyclohexane-N, N, N ′, N′-tetraacetic acid monohydrate) was added to the culture solution to 25 mM, and high performance liquid chromatography (used column Tosoh) TSKgel G5000PWXL) analysis gave a pectin concentration of 2.07 g / l and a cell growth rate of 42.8 times.

[Examples 3 and 4]
Culturing in the same manner as in Example 2 was carried out with a 300 ml medium amount of 500 ml and 800 ml with respect to a 3 liter flask to obtain pectin, respectively. The results are shown in Table 2.

[Example 5]
The cells induced in Example 1 were suspended in a liquid medium having the composition shown in Table 1 and cultured under the condition where the cell density was increased. The culture conditions were as follows: 40 ml of medium was added to a 300 ml flask, 25 ° C., dark place, cell density 200 g / l, rotary shaking culture at 100 rpm, and culture period 1 week. The culture solution was analyzed in the same manner as in Example 2. As a result, a pectin concentration of 1.51 g / l and a cell growth rate of 1.87 times were obtained.

[Examples 6 to 9]
In Example 5, the amount of the 40 ml medium was changed to 60, 80, 100 ml and 120 ml, and the same culture as in Example 5 was performed to obtain pectin. The results are shown in Table 3.

[Example 10]
A callus was induced in the same manner as in Example 1 and seedlings of Nigella sativa were cultured repeatedly 3 times to obtain a cell line that stably grew. The obtained cells were suspended in a liquid medium having the composition shown in Table 1 and cultured under the same conditions as in Example 7. Analysis of the culture medium gave a pectin concentration of 1.60 g / l and a cell growth rate of 18.6 times.

[Example 11]
40 ml of 0.5M ethylenediaminetetraacetic acid (pH 8) was added to 2 liters (5 flasks) obtained by removing solids such as cells from the culture solution obtained in Example 3 by filtration to solubilize pectin in the solution. After that, a finer solid was separated by filtration. 6 l of ethanol was added to the filtrate, and the mixture was gently stirred to obtain pectin as a precipitate. After refrigeration for 1 day, the precipitate was separated by filtration and redissolved in 1 l of water to obtain a pectin solution. Further, insoluble components were removed from the solution by filtration and freeze-dried to obtain 3.2 g of pectin as a white powder.

[Example 12]
(1) Preparation of calibration curve Polyethylene glycol (molecular weight 2 × 10 ⁶ , Wako Pure Chemical Reagent) was dissolved in water to a concentration of 1 g / l, and gel filtration chromatography was measured under the following conditions.

Column: Tosoh TSKgelG5000PWXL
Mobile phase: 0.1 M sodium nitrate aqueous solution Flow rate: 0.5 ml / min Temperature: 40 ° C.
Sample concentration: 1 g / l aqueous solution Sample injection volume: 10 μl
Detector: Differential refractometer (Shimadzu RID-10A)
In the obtained distribution curve, the retention time showing a peak was 10.8 minutes. Similarly, polyethylene oxide (molecular weight 1 × 10 ⁶ , 5 × 10 ⁵ , 2 × 10 ⁵ , GL Science Reagent) was measured by gel filtration chromatography, and the retention times showing peaks were 11.3 minutes and 11. 8 minutes and 13.2 minutes. A calibration curve was created by plotting the relationship between molecular weight and retention time.
(2) Measurement of molecular weight of pectin The pectin obtained in Example 11 was dissolved in water to a concentration of 1 g / l, and gel filtration chromatography was measured under the above conditions. The obtained distribution curve had a retention time in the range of 9.1 minutes to 13.2 minutes, and a retention time showing a peak of 10.8 minutes. From the calibration curve, the pectin obtained in Example 11 had a molecular weight range of 2 × 10 ⁵ to 1 × 10 ⁷ and a peak molecular weight of 2.0 × 10 ⁶ (FIG. 1).

[Test Example 1]
The pectin of the present invention obtained in Example 11 was examined for water retention ability by the paper disk method. That is, an aqueous solution with a concentration of 1 g / l of pectin of the present invention was prepared, 10 μl of a sample was impregnated into a filter paper having a diameter of 8 mm, and placed under conditions of a temperature of 25 ° C. and a relative humidity of 20%. Rate). As controls, water, 1 g / l sodium hyaluronate (Wako Pure Chemical, molecular weight range of 1 × 10 ^{5 to} 5 × 10 ⁶ , molecular weight peak 1.7 × 10 ⁶ ), aqueous solution, and 1 g / l A commercially available pectin having a concentration of l (citrus-derived pectin, Tokyo Kasei, molecular weight range of 1 × 10 ⁴ to 8 × 10 ⁵ and molecular weight peak of 1.0 × 10 ⁵ ) was used. The results are shown in Table 4. It was found that the pectin of the present invention has a high water retention rate and a high water retention capacity compared to the control.

[Test Example 2]
A 1 g / l aqueous solution of the pectin of the present invention obtained in Example 11 was prepared, and a biological stratum corneum water load test using a skin surface stratum corneum moisture measuring device SKICON-200 (IBS) was conducted. went. In the test method, in an environment of a temperature of 25 ° C. and a relative humidity of 20%, 20 μl of a sample was dropped on the skin on the inner side of the upper arm, wiped off after 10 seconds, and the moisture content of the skin horny layer thereafter was measured by a change in electrical conductivity. As a control, water used in Test Example 1 and a 1 g / l sodium hyaluronate aqueous solution and a 1 g / l commercial pectin aqueous solution were used. The results are shown in FIG. It can be seen that the pectin of the present invention has a higher ability to retain moisture on the skin than commercially available pectin and sodium hyaluronate, and is particularly excellent in skin permeability and affinity due to its high initial moisture content.

[Test Example 3]
A 1 g / l aqueous solution of the pectin of the present invention obtained in Example 11 was prepared, and the viscosity at a temperature of 35 ° C. and a stress of 1 Pa was measured using a rheometer (REOLOGICA). As a control, a 1 g / l aqueous solution of glycerin (Wako Pure Chemical Industries) and sodium hyaluronate (Wako Pure Chemical Industries) was used. The results are shown in Table 5. The pectin of the present invention gave a higher viscosity than glycerin, but was lower in viscosity than sodium hyaluronate. As a cosmetic material, this can provide a feeling of use with little stickiness while maintaining a moist and smooth feeling.

[Production Example 1]
0.1% by weight of pectin obtained in Example 11, 2.5% by weight of 1,3-butylene glycol, 0.5% by weight of glycerol (86%), 0.5% by weight of polyoxyethylene hydrogenated castor oil, lactic acid Lotion with a formulation of 0.05 wt%, sodium lactate 0.7 wt%, ethanol 7.0 wt%, methyl paraoxybenzoate 0.1 wt%, fragrance 0.05 wt%, purified water 88.5 wt% Was prepared.

[Production Example 2]
0.2% by weight of pectin obtained in Example 11, 4.0% by weight of liquid paraffin, 4.0% by weight of squalane, 0.5% by weight of cetanol, 1.5% by weight of stearic acid, sorbitan monooleate 0% by weight, polyoxyethylene sorbitan monooleate 1.0% by weight, glycerol monostearate 0.5% by weight, ethyl paraoxybenzoate 0.2% by weight, glycerol (86%) 3.0% by weight, 1, An emulsion was prepared with a formulation of 5.0% by weight of 3-butylene glycol, 0.05% by weight of fragrance, and 79.05% by weight of purified water.

[Production Example 3]
0.5% by weight of pectin obtained in Example 11, 8.0% by weight of petrolatum, 2.0% by weight of lanolin, 20.0% by weight of squalane, 5.0% by weight of cetanol, 2.0% by weight of glycerol monostearate %, Sorbitan polyoxyethylene monolaurate 2.0% by weight, ethyl paraoxybenzoate 0.2% by weight, glycerol (86%) 5.0% by weight, 1,3-butylene glycol 5.0% by weight, flavor 0. A cream was prepared with a formulation of 1 wt% purified water 50.2 wt%.

[Production Example 4]
Pectin 0.3% by weight, polyvinyl alcohol 18.0% by weight, polyethylene glycol 2.0% by weight, 1, 3 obtained by treating the culture solution obtained in Example 7 in the same manner as in Example 11. -A pack was prepared with a formulation of 5.0% by weight of butylene glycol, 8.0% by weight of ethanol, 0.1% by weight of methyl paraoxybenzoate, 0.05% by weight of fragrance and 66.55% by weight of purified water.

[Production Example 5]
15. Pectin 1.0% by weight, 1,3-butylene glycol 20.0% by weight, glycerol (86%) obtained by treating the culture solution obtained in Example 10 in the same manner as in Example 11. 0% by weight, polyethylene glycol 5.0% by weight, polyoxyethylene hexadecyl ether 0.1% by weight, citric acid 0.05% by weight, sodium citrate 0.5% by weight, methyl paraoxybenzoate 0.2% by weight Essence was prepared according to a prescription of 0.1% by weight of fragrance and 58.05% by weight of purified water.

[Production Example 6]
10. Pectin 0.3% by weight, talc 30.0% by weight, sericite 25.0% by weight, titanium oxide 10% obtained by treating the culture solution obtained in Example 7 in the same manner as in Example 11. 0% by weight, modified silicone 7.0% by weight, polyethylene powder 4.0% by weight, squalane 2.0% by weight, methyl paraoxybenzoate 0.1% by weight, fragrance 0.05% by weight, purified water 21.55% by weight A foundation was prepared with a% formulation.

[Production Example 7]
0.3% by weight of pectin, 45.0% by weight of modified silicone, 20.0% by weight of candelilla wax, paraffin wax obtained by treating the culture solution obtained in Example 10 in the same manner as in Example 11. 10.0% by weight, carbana wax 10.0% by weight, isobutyl isopalmitate 4.0% by weight, diisostearyl malate 3.7% by weight, isononyl isononanoyl 2.0% by weight, red No. 2022. Lipsticks were prepared according to the formulation of 0% by weight, Red No. 101 1.0% by weight, Titanium oxide 1.0% by weight, Yellow No. 4 1.0% by weight.

  As detailed in the Examples, high molecular weight pectin can be provided by cell culture of the plant of the present invention. Since the obtained high molecular weight pectin has a high water retention ability and low viscosity, it can be used as a cosmetic material, particularly as a moisturizing agent.

Claims (5)

It contains galacturonic acid as a constituent sugar in an amount of 70 to 90 mol%, a molecular weight range measured by gel filtration chromatography is 1 × 10 ^{4 to} 5 × 10 ⁷ , and a molecular weight exhibiting a peak is 2 × 10 ⁵ to A method for producing pectin, comprising obtaining and recovering pectin in the range of 1 × 10 ^{7 from} a culture or culture solution obtained by culturing cells of the genus Nigella.   The molar ratio of the constituent sugars of the pectin is galacturonic acid: arabinose: galactose: glucuronic acid: glucose: xylose: rhamnose: mannose: fructose = 7-9: 0.4-0.7: 0.4-0.6: It is 0.1-0.6: 0.05-0.5: 0.04-0.4: 0-0.0.1: 0-0.08: 0-0.05. The method for producing pectin according to 1.   The method for producing pectin according to claim 1 or 2, wherein the plant of the genus Nigella is Nigella damascena or Nigella sativa. It contains galacturonic acid as a constituent sugar in an amount of 70 to 90 mol%, a molecular weight range measured by gel filtration chromatography is 1 × 10 ^{4 to} 5 × 10 ⁷ , and a molecular weight exhibiting a peak is 2 × 10 ⁵ to Separating and recovering pectin in the range of 1 × 10 ⁷ from a culture or culture obtained by culturing cells of the genus Nigella,
A method for producing a humectant comprising blending the pectin separated and recovered from the culture or the culture as an active ingredient . It contains galacturonic acid as a constituent sugar in an amount of 70 to 90 mol%, a molecular weight range measured by gel filtration chromatography is 1 × 10 ^{4 to} 5 × 10 ⁷ , and a molecular weight exhibiting a peak is 2 × 10 ⁵ to Separating and recovering pectin in the range of 1 × 10 ⁷ from a culture or culture obtained by culturing cells of the genus Nigella,
A method for producing a cosmetic comprising blending the pectin separated and recovered from the culture or the culture solution as an active ingredient of a moisturizing agent .